Expression of a Plant Sesquiterpene Cyclase Gene in Escherichia coli

Back, Kyoungwhan; Yin, Shaohui; Chappell, Joe

doi:10.1006/abbi.1994.1533

Cited by 54 publications

(49 citation statements)

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“…2B), values consistent with our previous characterization of these products as 5-epi-aristolochene and vetispiradiene, respectively (41,43). Additional analysis by GC and GC-MS indicated that the predominant TEAS reaction products were 5-epi-aristolochene (70% of total products, based on percentage of total peak areas from GC analysis; ref.…”

Section: Resultssupporting

confidence: 88%

“…CHs were generated by substituting the corresponding domains between the tobacco and Hyoscyamus genes, the chimeric genes were expressed in bacteria, and bacterial lysates were assayed for sesquiterpene cyclase activity (43). The sesquiterpene cyclase assays included an argentation-TLC analysis that easily distinguishes the TEAS-specific and HVS-specific products from one another (41).…”

Section: Resultsmentioning

confidence: 99%

“…Escherichia coli TB1 cells were used for all the expression studies. Procedures for growth of the bacterial cells, induction of gene expression, and measurement of sesquiterpene cyclase enzyme activity and total protein in the bacterial lysates were identical with those described (41,43). The predominant reaction products were separated from one another by developing G60 silica TLC plates impregnated with 15% silver nitrate in benzene:hexane:diethyl ether (50:50:1).…”

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confidence: 99%

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Identifying functional domains within terpene cyclases using a domain-swapping strategy.

Back

Chappell

1996

Proc. Natl. Acad. Sci. U.S.A.

106

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Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plantenvironment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes The biosynthesis of cyclic terpenes is determined by key branch point enzymes referred to as terpene cyclases, or more properly, terpene synthases. The reactions catalyzed by terpene cyclases are complex, intramolecular cyclizations that may involve several partial reactions (8, 9). For example, the bioorganic rationale for the cyclization of farnesyl diphosphate by two sesquiterpene cyclases pertinent to this work are illustrated in Fig. LA. In step 1, the initial ionization of farnesyl diphosphate is followed by an intramolecular electrophilic attack between the carbon bearing the diphosphate moiety and the distal double bond to form germacrene A, a macrocyclic intermediate. Internal ring closure and formation of the eudesmane carbonium ion constitutes step 2. For tobacco 5-epi-aristolochene synthase (TEAS), the terminal step is a hydride shift, methyl migration, and deprotonation at C9, giving rise to 5-epi-aristolochene (step 3a). Hyoscyamus muticus vetispiradiene synthase (HVS) shares common mechanisms at steps 1 and 2, but differs from TEAS in the third partial reaction, in which a ring contraction would occur due to alternative migration of an electron pair. In each case, a monomeric protein of -64 kDa catalyzes the complete set of partial reactions and requires no cofactors other than Mg2+.Numerous terpene cyclases from plant and microbial sources have been partially or completely purified and characterized (11-22). These enzymes behave as soluble enzymes with molecular weights of 40,000-100,000. Mechanistic studies have included evaluations of the proposed reaction mechanisms (23-27), efficacy of substrate analogs (28, 29) and suicide inhibitors (28)(29)(30), and the use of chemical-modifying reagents and site-directed mutagenesis to identify amino acids essential for catalysis (31)(32)(33). Recently, a number of fungal and plant genes for monoterpene, sesquiterpene, and diterpene cyclases have been described (34-41). The plant monoterpene, sesquiterpene, and diterpene cyclases exhibit a significant degree of similarity at the amino acid level, and at least for the sesquiterpene and diterpene cyclases, the intron/exon organization of genomic DNA is nearly identical (40-42). In contrast, other than perhaps the conservation of a 5-aa sequence rich in aspartate residues, very little similarity is observed between the fungal and plant sesquiterpene cyclase proteins (38).One implication of the sequence similarity and conserved intron/exon organization observed between the plant genes is that regions of sequence conservation may correspond to functional domains, and those functional domains may mediate the catalysis of particular partial reactions. This inference was recently teste...

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Identifying functional domains within terpene cyclases using a domain-swapping strategy.

Back

Chappell

1996

Proc. Natl. Acad. Sci. U.S.A.

106

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“…Protein expression assays were essentially as described in Back et al (1994) and Back and Chappell (1995). Liquid cultures of the clones in 250 mL of Luria broth were grown to an OD 600 of 1.0.…”

Section: Nuclear Magnetic Resonancementioning

confidence: 99%

Genetic Control and Evolution of Sesquiterpene Biosynthesis in Lycopersicon esculentum and L. hirsutum

et al. 2000

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Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 ( Sst1 ), was identified for which the L. esculentum allele is associated with the biosynthesis of ␤ -caryophyllene and ␣ -humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B , and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2 , on chromosome 8, the L. hirsutum allele specified accumulation of ␣ -santalene, ␣ -bergamotene, and ␤ -bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.

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“…The Protox expression construct was prepared by inserting the full-length cDNA without a stop codon into the pET28(b) bacterial expression vector. 14,15) The insert was subcloned in frame so that a hexa-histidine tag was added to the carboxy terminus of the protein. The expression of recombinant Protox is highly induced by IPTG.…”

Section: Methodsmentioning

confidence: 99%

Either Soluble or Plastidic Expression of Recombinant Protoporphyrinogen Oxidase Modulates Tetrapyrrole Biosynthesis and Photosynthetic Efficiency in Transgenic Rice

Jung¹,

Chung

Jang

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Expression of a Plant Sesquiterpene Cyclase Gene in Escherichia coli

Cited by 54 publications

References 0 publications

Identifying functional domains within terpene cyclases using a domain-swapping strategy.

Identifying functional domains within terpene cyclases using a domain-swapping strategy.

Genetic Control and Evolution of Sesquiterpene Biosynthesis in Lycopersicon esculentum and L. hirsutum

Either Soluble or Plastidic Expression of Recombinant Protoporphyrinogen Oxidase Modulates Tetrapyrrole Biosynthesis and Photosynthetic Efficiency in Transgenic Rice

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